Functional characterization of the zebrafish genes requires accurate knowledge of the gene structure and gene expression. In proposal we combine high-throughput sequencing techniques (Ribosome profile and RNA-seq) and bioinformatics to improve the current annotation of coding and non-coding genes, by determining the translation start and stop sites in each coding gene (Aim 2), define the transcription start site, 5' and 3'UTRs (Aim 1), annotate putative alternatively spliced exons in the zebrafish genes (Aim 1), and develop a gene expression atlas during embryogenesis and organogenesis (Aim 1) The experiments outlined in this proposal have the ultimate goal of improving functional genomics in zebrafish and will provide two fundamental tools to the community: i) improved annotation of the zebrafish genes including coding potential, translation start site and transcript structure and ii) community defined, open access zebrafish expression atlas across embryonic/ larval stages and organogenesis. In the future, the results derived from this project will facilitate the functional characterization of the zebrafish genes and will establsh a frame work to understand the structure and the function of vertebrate genes. PUBLIC HEALTH RELEVANCE: The genome of vertebrate animals is very similar to that of humans. By studying the secrets underlying in the genetic code of the vertebrate model system zebrafish, we aim to characterize the elements of the genome to provide better tools to zebrafish researchers to understand vertebrate development and human disease. The experiments outlined in this proposal will also help us uncover novel genes unknown in humans providing important insights about the building blocks that are used in humans to undertake different functions in the cell.